Lung cancer is the leading cause of cancer related deaths in the USA. The most frequent form of lung cancer is non small cell lung cancer (NSCLC). Among the three major histological types of NSCLC, adenocarcinoma of the lung (ACL) is the most prevalent, and its incidence is steadily raising. ACL is relatively common (from 15 to 20% of cases) in people who have never smoked, therefore ACL will remain a major health problem even after cigarette smoking eradication. ACL is normally diagnosed at advanced stages because early detection methods remain problematic. Median survival after advanced ACL diagnosis is only 5 months, and chemotherapy only slightly improves it. It is therefore imperative to find novel therapeutic strategies to treat this deadly disease. Our preliminary data indicate that the evolutionarily conserved Notch-1 receptor provides critical survival signals to ACL cells in hypoxia, a condition that best recapitulates ACL microenvironment. Hypoxic conditions are those that interfere with standard chemotherapy and favor the preservation of niches for cancer stem cells. Therefore, targeting a signaling pathway that favor survival of cancer cells in hypoxia represents a particularly promising therapeutic strategy for the treatment of ACL. To be activated, Notch-1 requires a number of proteolitic cleavages, including the participation of a ?-secretase. Since this protein also participates in the accumulation of -amyloid in Alzheimer's Disease, a great effort has been spent to synthesize specific small molecules that inhibit ?-secretase (?-secretase inhibitors or GSI). Therefore, GSI are indirect inhibitors of Notch signaling because, in the absence of the activating proteolitic cleavage catalyzed by ?-secretase, Notch-1 (as well as other Notch receptors) cannot translocate in the nucleus and affect transcription. We used as GSI a compound developed by Merck (MRK-003). This compound proved very efficient in killing ACL cells specifically under hypoxia. Our hypothesis is that Notch-1 provides survival signals to ACL cells mediating an intricate network of signaling pathways that includes PTEN, STAT, IGF-1, IGF-1R, Akt and its downstream targets. We also hypothesize that targeting Notch signaling in vivo is a valid therapeutic strategy for the treatment of ACL. We want to verify our hypotheses according to the following Specific Aims: (1) Verify whether there is interplay and a positive feedback loop between Notch-1 and the IGF-1 signaling pathway and what are the biological consequences of such interaction in hypoxia. (2) Study the molecular mechanisms through which hypoxia strengthen Notch-1 signaling and how Notch-1 protects ACL cells from apoptosis specifically under hypoxia. We will focus on Notch-1 regulation of PTEN and the PI3 kinase/PDK-1/Akt/mTOR axis. (3) Verify the therapeutic efficacy of MRK-003, alone or in combination with cisplatin, or with an inhibitory antibody for the IGF-1R (MK-0646), or with an Akt specific inhibitor (MK-2206) in a mouse preclinical model of advanced ACL. We will use cisplatin in combination with MRK-003 to simultaneously target non-hypoxic and hypoxic tumor areas. MK-0646 and MK-2206 will be also used in separate experiments because these compounds may have synergistic effects with MRK-003 because of our preliminary results sustaining Aims 1 and 2. We will dissect the contribution of Notch inhibition mechanistically using ACL cell lines in which we can artificially downregulate Notch-1. The results obtained in Aim 1 and 2 will be verified in vivo in the mouse model and in human clinical samples. The outcome of the research proposed here will provide a better understanding of the molecular processes that contribute to the maintenance of the malignant phenotype in ACL. Furthermore, the study will test the clinical usefulness of targeting Notch signaling to treat NSCLC using GSI. Finally, these experiments will elucidate the role of Notch-1 in lung tumorigenicity in vitro and in vivo.